Circadian clocks are intracellular timekeeping mechanisms that allow cells to coordinate various aspects of their physiology with time of day. The central feature of this circadian clock is a core transcriptional/translational negative feedback loop. The many rhythmic "outputs" of the clock are controlled by coordinate regulation of many other genes downstream of the central clock genes. Studies on this mechanism have focused on transcriptional and post-translational mechanisms, however, there is significant evidence that post-transcriptional mechanisms must also be involved in generating stable circadian rhythms. The best candidate for mediating circadian post-transcriptional control in vertebrates is the protein Nocturnin (NOC), a deadenylase (an enzyme that removes the polyA tail from mRNAs) that is expressed in clock- containing cells with high amplitude rhythms. Our hypothesis is that NOC contributes to circadian regulation through its control of the half-life (or translatability) of specific rhythmic mRNAs. We have recently discovered that loss of the Noc gene in mice results in resistance to diet-induced obesity. In this revised proposal, we extend experiments proposed in the original proposal on the biochemical function of NOC into newly generated mouse models that will provide insight on the metabolic changes that are regulated by NOC. This involves adding additional in vivo experiments into our previous Specific Aims and adding a new Specific Aim 5 focused on metabolism. Therefore, the aims in this revised application are as follows: (1) Characterization of the complex of proteins in which NOC resides; (2) Examine NOC's intracellular localization; (3) Determine whether NOC deadenylates its own mRNA to form an autoregulatory feedback loop; (4) Identify NOC's target mRNAs and determine how these targets are recognized; and (5) Characterization of the mechanism that causes the lean phenotype in the Noc KO mice. The studies proposed here will contribute to the understanding of the molecular underpinnings of these clocks and to the general area of post-transcriptional control of gene expression. In addition, these experiments will produce new information about how the circadian system controls obesity. PUBLIC HEALTH RELEVANCE: Circadian clocks control many aspects of physiology and behavior and disruption of circadian rhythmicity has been linked to many medical disorders including cancer, sleep problems and obesity and diabetes. The proposed work will address how the circadian gene Nocturnin contributes to the proper control of metabolic processes in mammals.